N
TOMÁŠKŘÍŽEKa, PAVEL OUFALC a, RADOMÍR ABALAČ a, HELENA YŠLAVÁR b
aDepartment of Analytical Chemistry, Department of Biochemistry, Faculty of Science, Charles University in Prague,b
Albertov6/2030, 128 43 Prague, Czech Republic,*krizek@natur.cuni.cz
Keywords
Study of enzyme kinetics of glycosidases is inevitable for understanding their functions in complex biological systems. However, such a study cannot be performed without monitoring the concentration of products and substrates of these enzymes. For this purpose, a fast and reliable analytical method is necessary. The separation mechanism of capillary electrophoresis is well suited for separation of chitobiose and -acetyl glucosamine as a substrate and product of - -acetylhexosaminidase. Thus a reliable and sufficiently sensitive capillary electrophoresis method for their determination has been developed and successfully utilized for kinetics studies of - -acetylhexosaminidase.
Reliability of the method was confirmed by repeatability studies and chemometric analysis of calibration data. A remarkable advantage of the method lays in no need of derivatization of analytes as well as the fact that the enzyme reaction is terminated simply by injecting the reaction mixture into the capillary.
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Glycosidases are enzymes that catalyze cleavage of poly- and oligosaccharide chains. Expression of some glycosidases is also connected with pathogenesis that takes place under stressing conditions [1]. - -acetyl hexosaminidase is a representant of glycosidases enzyme family and catalyzes for example cleavage of disaccharide chitobiose to monosaccharide -acetyl glucosamine. - -acetylhexosaminidase has several functions in the biochemistry of fungi [2 4]. The investigation of activity and kinetics of this enzyme is crucial for understanding the complex biological processes in which - -acetylhexosaminidase parti cipates. The activity of enzymes is usually studied by observing the concentration of their substrates and products in the reaction mixture. Such a procedure, however, requires a fast analytical method of a suffi cient sensitivity and selectivity.
In the literature, mainly separation techniques were reported for determination of saccharides [6].
Capillary electrophoresis (CE) is well established as a tool for determination of saccharides [7, 8]. However, the CE separation of saccharides is rather complicated problem as they possess electric charge only in highly alkaline solutions and this charge is negative which means that they exhibit counter-electroosmotic flow migration resulting in a long analysis time, enhanced adverse dispersion effects and a lower repeatability of
β N
measurements. Moreover, saccharides exhibit very low UV absorbance. Both these difficulties can be solved by derivatization of analytes before analysis.
Appropriate chromophore [9] or fluorophore [10] can thus be introduced into the molecule of analyte. This approach enables highly sensitive detection, espe cially when the fluorescence detector is used. On the other hand, the derivatization brings the risk of sample loss or contamination during the procedure as well as a prolonged sample processing. Therefore, methods using indirect UV [11] or conductivity detection [12]
have been developed. Nevertheless, none of them surpassed the others remarkably. In this work, a method for separation of saccharides chitobiose and -acetylglucosamine was developed. As can be seen in Figure 1, these analytes contain the UV absorbing amide bond in their structures and thus direct UV detection can be utilized with sufficient sensitivity.
-N
2. Experimental
3. Results and Discussion 2.1. Chemicals
2.2. Instrumentation
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β- -acetylhexosaminidase was graciously donated by Prof. Karel Bezouška (Charles University in Prague, Faculty of Science, Department of Biochemistry).
The enzyme was isolated from
strain CCF1066 following the procedure described in cit. [13]. Sodium hydroxide, p.a., and sodium tetra borate decahydrate, p.a., were purchased from Lachema (Brno, Czech Republic). -diacetyl chitobiose,
-≥ 96%, and -acetyl- -glucosamine,
≥ 99%, were delivered by Sigma (St. Louis, MO, USA). Background electrolytes and samples were prepared using deionized water produced by a Milli-Q system, Millipore (Billerica, MA, USA).
N D
Experiments were carried on an Agilent CE3D
apillary. Prior to the first use, the capillary was flushed 20 min with 1 M sodium hydroxide and 10 min with water using a pressure of 100 kPa. Before each run, the capillary was flushed 2 minutes with background electrolyte. Samples were injected using ei
Chitobiose is a dimer of -acetylglucosamine as obvious from the Figure 1. Since the electrophoretic instrument (Agilent Technologies, Waldbronn, Ger7200 many). Fused silica capillaries, 50 and 75 μm i d, were cut to the 65 cm length. Detection window was burnt by a butane flame 56.5 cm from the inlet end of the c
ther pressure of 5 kPa for 3 s or 5 kV for 5 s.
A voltage of 15 kV was applied during the separation (30 kV for 50 μm id). The capillary was thermostated at 25 °C.
-.
mobility is proportional to the ratio of charge and hydrated radius of an ion, chitobiose and -acetyl glucosamine differ in their electrophoretic mobilities which means that, despite their similar chemical properties, they can be separated by CE. Saccharides are ionized (and migrate in electric field) only in extre mely alkaline background electrolytes. However, these electrolytes possess a critically high conduc tivity, which makes them rather unsuitable for CE experiments. To avoid use of highly alkaline electro lytes, borate buffer was employed as the borate anions form negatively charged complexes with saccharides [14, 15]. These complexes then migrate in electric field and can be separated. As 20 mM sodium tetraborate buffer, pH 9.2, did not provide baseline separation of the analytes, ionic strength was elevated to 25 mmol L . The mentioned buffer provided a complete separation and the current of 42 μA was still acceptable with respect to Joule heat produced during the run.
The method was originally developed for 50 μm i d capillary and provided separation in 5 minutes.
However, for the quantification purposes, 50 μm i d capillary was found to be unsuitable because the repeatability of peak area was insufficient. This can be attributed to a higher tendency of the 50 μm i d capillary to clog. Due to instable hydrodynamic resis tance in capillary, the efficiency of sample injection varied and the peak area became irreproducible. Thus the method was adapted to 75 μm i d capillary. As the higher inner diameter brings a significantly higher electric current, the voltage had to be reduced to 15 kV so that the corresponding current of 55 μA was kept at acceptable level. This voltage adjustment prolonged the analysis time to 9 minutes. Nevertheless, Table 1 shows that a three-fold improvement of the peak area repeatability was achieved. Electrokinetic and hydro dynamic injection modes were tested and are compa red in Table 1 as well. Based on the results, the
N
relative standard deviation [%]
50 m i d capillary 75 m i d capillary
time area height time area height
Hydrodynamic injection
Chitobiose 0.7 14.1 11.1 1.4 8.1 5.6
-Acetylglucosamine 0.8 15.2 15.5 1.4 6.5 4.5
Electrokinetic injection
Chitobiose 0.4 11.5 9.3 1.6 3.4 4.6
-Acetylglucosamine 0.4 11.8 12.5 0.8 3.4 3.5
μ μ
N
N Table 1
The repeatability of migration times, peak areas and peak heights in 50 and 75 m i d capillaries expressed as the relative standard deviation, = 10. B : 25 mM sodium tetraborate, pH 9.2; temperature 25 °C; voltage 30 kV for 50 m and 15 kV for 75 m capillary; detection at 200 nm; concentration 1 10 mol L .
μ μ μ
ackground electrolyte. .
= ×
n
–3 –1
Fig. .2 Separations of chitobiose (1) and -acetylglucosamine (2) in capillaries of the 50 and 75 m inner diameter. B
: 25 mM sodium tetraborate, pH 9.2; temperature 25 °C, voltage 30 kV for 50 m and 15 kV for 75 m capillary, detection at 200 nm, concentration 1×10 mol L .
N
μ ackground
electrolyte =
μ μ
–3 –1
electrokinetic injection was chosen because it provided a higher repeatability (see Table 1).
Electropherograms obtained with the 50 and 75 μm i d capillaries are displayed in Figure 2.
The 75 μm i d capillary was used for further experiments. Calibration standards in the concen tration range from 0.1 to 2.0 mmo were measured and linear regression was performed using the least squares method. The resulting calibration curve was subjected to chemometric analysis. Parameters of the method for determination of chitobiose and -acetyl glucosamine are shown in Table 2. Obviously, linear regression provides a satisfactory model of response as it explains 99.7% of the response variability for both analytes (see values). The deviation of line arity coefficients from ideal value, = 1, is safely within 5% range, which means that the detector res ponse can be considered as linear over the studied
. . . .
l
-L–1
N
R
l
2
concentration range. These results show that an elevation of the baseline after the chitobiose peak observed in electropherograms with the 75 μm capillary has no adverse effect on quantification. The limit of detection and the limit of quantification were calculated from standard deviation of intercept; their values 0.4 10 and 1.2 10 mol , respectively, show that the developed method is sufficiently sen sitive for the enzyme kinetics study.
The developed method was employed to prove that chitobiose is the substrate of - -acetylhexos aminidase. Figure 3 shows a decrease of the chitobiose concentration and an increase of the -acetylglucosamine concentration in the reaction mixture. The graph in the Figure 3 also suggests that one molecule of chitobiose is cleaved into two molecules of -acetylglucosamine which is in accor dance with the expected stoichiometry. The developed
× ×
-–4 –4 L–1
-β N
N
N
Fig. 3. Concetrations of chitobise (♦) and -acetylglucos-amine (◊) in the reaction mixture as a function of the reaction time.
The enzyme hexosaminidase diluted 1/20 000. 50 mM citrate buffer, pH = 4.5.
N
Chitobiose -Acetylglucosamine Calibration range 0.1–2.0 mmol L 0.1 2 mmol
Slope 21.05 min L mmol 51.44 min L mmol
Intercept 0.2682 min 0.4182 min
0.9975 0.9972
27 27
Linearity coefficient 0.9750 0.9968 Limit of detection 0.04 mmol 0.04 mmol Limit of quantification 0.12 mmol 0.12 mmol
N
R N
–1
2
– .0 L
– –
L L
L L
–1
–1 –1
–1 –1
–1 –1
Table 2
The calibration parameters of the capillary electrophoresis method for determination of chitobise and -acetylglucosamine.N
method is also advantageous for the kinetics study because the enzymatic reaction can be terminated simply by injection of the reaction mixture into the capillary. The enzymatic reaction stops immediately after beginning of the run because the overall charge of the enzyme is under the employed conditions positive and the enzyme and the chitobiose molecules migrate in the opposite direction.
A capillary electrophoresis method for fast simul taneous determination of chitobiose and -acetyl glucosamine as the substrate and product of the enzyme - -acetylhexosaminidase has been deve loped. The reliability of the method was confirmed by the migration time and peak area repeatability study.
Further, the evaluation of the calibration curve proved that the method is well applicable for quantification over the concentration range considered. The method was used to prove that - -acetylhexosaminidase catalyzes the cleavage of chitobiose into two molecules of -acetylglucosamine. A study of - -acetylhexosaminidase kinetics is in progress and is to be followed by an enzyme kinetics study with chitotriose, which is a trimer of -acetylglucosamine.
4. Conclusions
The financial support of the Grant Agency of Charles University in Prague, projects No. 710 and SVV 261204, the Ministry of Edu cation, Youth and Sports of the Czech Republic, project MSM0021620857 and RP 14/63 as well as the Norwegian Financial Mechanism, project CZ0116, is gratefully acknow ledged.
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